Multiplex Immunofluorescence on Formalin-Fixed Paraffin-Embedded Mouse Tissue (Version 1.0)
Version
1.0
Notice
This page is the corresponding protocol tomestone page generated as part of the ATLAS-D2K shutdown in July 2025. Many links on this page may be broken.
Authors
Mark de Caestecker; Maya Brewer
Keywords
[‘antibodies’, ‘immunofluorescence’, ‘kidney’, ‘mouse’]
Subjects
[‘Imaging’, ‘Immunological techniques’]
Release Date
2022-03-23
Abstract
To describe the procedure for multiple cycles of immunofluorescence on formalin-fixed paraffin-embedded mouse kidney tissue. Kidney sections are tagged with fluorescent-labeled antibodies and imaged. The fluorophore is then deactivated followed by the addition of a new set of directly conjugated antibodies.
Reagents
• Citrisolv/Xylene • 100% Ethanol • ddH2O • 10X Target Retrieval Solution, Citrate pH 6.1 (Agilent, S169984-2) • 1X Phosphate Buffered Saline (PBS) • Triton X-100 (Sigma-Aldrich, T8787) • Hydrophobic Barrier Pen (Vector Labs, H-4000) • Glycine (Sigma-Aldrich, 410225) • Avidin/Biotin Blocking Kit (Vector Labs, SP-2001) • 10X Power Block Universal Blocking Reagent (BioGenex, HK085-5K) • Antibody Diluent Reagent Solution (Thermo Fisher, 003218) • Hoechst 33342 (20mM, Thermo Fisher, 62249) • 100% Glycerol (Fisher Scientific, G33) • 30% Hydrogen Peroxide (Sigma-Aldrich, 216763) • 1 M Sodium Hydroxide (Fisher Scientific, SS266-1)
Equipment
• Steamer for antigen retrieval • Microwave or pressure cooker can be used • Moisture Chamber (see Figure 1) - 100-Slide Storage Box (Fisher Scientific, 03-448-1) - Kimwipes, 8.4 in x 4.4 in (Fisher Scientific, 06-666) - ddH2O in a wash bottle • LED Cabinet Light (see Figure 2) - (Sears, SPM11582738325) • Square Cell Culture Plate
Procedure
De-Paraffinization and antigen retrieval (FFPE tissue only)
1) De-wax and hydrate paraffin sections. 2) Transfer slides to 1X PBS. 3) Dilute 10X citrate buffer to 1X using ddH2O. 4) Place slides in buffer and cap the container holding the slides. Steam for 26 minutes. 5) Remove slides from steamer. Remove the cap and allow slides to equilibrate to room temperature. 6) Pour citrate buffer into appropriate waste container and wash slides in 1X PBS for 5 minutes three times.
I) Immunofluorescence
1) Using a hydrophobic pen, draw a large barrier around each section. Do not allow pen to touch section. 2) Place slides in humidified chamber and cover section in PBS by using a pipettor so that they do not dry. a) To remove solutions from sections that are in the humidified chamber, tip the solution off of the slide into the chamber (see Figure 1 below). 3) Optional: Incubate sections for 5 minutes with 50 mM glycine (dilute stock in 1X PBS). This reduces autofluorescence by reducing free aldehyde groups. Remove glycine, and wash sections in PBS for 5 minutes twice. 4) If using Biotin Amplification (Optional): a) Block sections in avidin solution for 15 minutes. b) Wash sections in PBS twice. c) Block sections in biotin solution for 15 minutes. d) Wash sections in PBS twice. 5) Block sections for 30 minutes with 1X Universal Blocking Reagent (UBR) at room temperature. a) Dilute 10X blocking reagent to 1X using 9-parts ddH2O to 1-part UBR. 6) Dilute primary antibody to desired working concentration in Antibody Diluent Reagent during blocking step. 7) Add diluted antibody to section and incubate overnight at 4˚C. 8) Remove solution, and wash sections with 0.1% Triton X-100 in PBS (PBST) for 5 minutes three times. 9) If primary antibody is directly conjugated with a fluorophore, skip to #14. 10) If using indirect immunofluorescence, dilute fluorophore-conjugated secondary antibody using Antibody Diluent Reagent. 11) Add antibody solution to sections and incubate for 60 minutes at room temperature. 12) Remove solution, and wash sections with PBST for 5 minutes three times. 13) If a biotin-conjugated primary or secondary antibody was used: a) Dilute fluorophore-conjugated Neutravidin in PBS at 1:250 for 30 minutes. b) Wash sections in PBS for 5 minutes three times. 14) Incubate sections in Hoechst 33342 (1:5,000 dilution of 20mM solution in 1X PBS) for 5 minutes. 15) Remove Hoechst 33342, and wash sections with PBS for 5 minutes twice. 16) Mount slides in 50% glycerol in PBS. Do not seal the coverslips as they will need to be removed later (note that because they are not sealed, slides have to be kept horizontal, to prevent the coverslip from falling off, and in the humidified chamber) 17) Image 18) Store slides at 4˚C in a moisture chamber.
II) De-Coverslipping 1) Remove coverslip from sections by incubating slides in a vertical staining jar filled with PBS for 15-30 minutes with slight agitation (i.e. plate rocker). 2) Slowly lift slide from vertical jar and allow coverslip to release from slide via gravity. 3) Wash slide in PBS for 5 minutes three times to remove any residual glycerol. a) Place slides back in vertical staining jar full of PBS with slight agitation to wash. III) Fluorophore inactivation 1) Make a solution of 4.5% hydrogen peroxide and 24 mM sodium hydroxide made up in PBS. 2) Add bleaching solution to each section and incubate at room temperature for 90-120 minutes in the presence of white light (LED light). For this, place slides on a plastic surface on top of the LED light. We often use multiple plates for this (See Figures 2) a) Solution is removed and replaced with fresh solution every 30 minutes to ensure complete inactivation occurs. 3) Remove solution and wash sections in PBS for 5 minutes four times. a) After inactivating the fluorophores, slides are mounted in 50% glycerol and imaged to confirm complete fluorophore inactivation, followed by the removal of the coverslip, and three 5 minutes washes in PBS. Hoechst stain will not bleach and is necessary for image registration later
IV) Subsequent Immunofluorescence Cycles 1) Dilute fluorophore-conjugated primary antibodies using Antibody Diluent Reagent. 2) Add diluted antibodies to each section and incubate overnight at 4˚C. 3) Remove antibody solution and wash sections with PBST for 5 minutes three times. 4) Incubate sections in Hoechst (1:10,000 dilution in 1X PBS) for 5 minutes (this may not be necessary, but does no harm) 5) Remove Hoechst, and wash sections with PBS for 5 minutes twice. 6) Mount slides in 50% glycerol in PBS. 7) Image 8) Store slides at 4˚C in a moisture chamber.
Steps II-IV are repeated for each remaining cycle.
References
- Lin JR, Izar B, Wang S, Yapp C, Mei S, Shah PM, Santagata S and Sorger PK. Highly multiplexed immunofluorescence imaging of human tissues and tumors using t-CyCIF and conventional optical microscopes. Elife. 2018;7.
Consortium
(Re)Building a Kidney (RBK) Consortium
